1. Field of the Invention (Technical Field)
This invention relates to peptide-based metal ion-labeled compositions for use as pharmaceuticals, and methods of labeling peptides, proteins and other similar substances with radiometals, paramagnetic metals and other medically useful metal ions, and further providing for use of medically useful metal ion-labeled peptides for detection of thrombus, cancer, infection and inflammation.
2. Description of the Related Art, Including Information Disclosed under 37 C.F.R. Sections 1.97-1.99 (Background Art)
The use of proteins, particularly antibodies, as biologically active targeting agents for medically useful metal ions has been explored. These products can be administered to the human body to visualize or monitor functioning of various parts of the body or to determine the presence and location of particular antigens, antibodies, hormones or the like; and can be used in the treatment of various disease states. Antibodies and antibody fragments have been labeled with a number of radionuclides for use in clinical diagnosis. Radionuclides commonly used include .sup.131 I, .sup.125 I, .sup.123 I, .sup.99m Tc, .sup.67 Ga, and .sup.111 In for diagnostic imaging; and radionuclides such as .sup.90 Y, .sup.188 Re, and .sup.186 Re, and to a lesser extent, .sup.199 Au, .sup.131 I and .sup.67 Cu for targeted therapy, primarily in the treatment of cancer. There are also useful metals for magnetic resonance imaging, including gadolinium, manganese, copper, iron, gold and europium, which are not radioisotopes. So far, limited work have been done with labeling with positron-emitting radiometals, although some types of proteins, such as transferrin and human serum albumin, have been labeled with .sup.68 Ga.
Two primary methods have been employed to label antibodies with radiometals, with particular emphasis having been placed on radiolabeling with .sup.99m Tc. In one method, bifunctional chelates are conjugated to the antibody, and the bifunctional chelate is then radiolabeled. A variety of bifunctional chelates have been employed; most involve metal ion binding to thiolate groups, and may also involve metal ion binding to amide, amine or carboxylate groups. Representative bifunctional chelates include ethylenediamine tetraacetic acid (EDTA), diethylenetetramine-pentaacedic acid (DTPA), chelates of diamide-dimercaptides (N.sub.2 S.sub.2), and variations on the foregoing, such as chelating compounds incorporating N.sub.2 S.sub.3, N.sub.2 S.sub.4 or N.sub.3 S.sub.3 metal binding sites, and metallothionine. The alternative method of radiolabeling antibodies involves reduction of disulfide bonds in the protein, with subsequent binding of the metal ion to thiolate groups. A variety of reducing agents have been employed, including stannous salts, dithiothreitol and 2-mercaptoethanol.
The use of biologically active peptides, which are peptides which bind to specific cell surface receptors, has received some consideration as radiopharmaceuticals. Canadian Patent Application 2,016,235, Labeled Chemotactic Peptides to Image Focal Sites of Infection or Inflammation, teaches a method of detecting a site of infection or inflammation, and a method for treating such infection or inflammation, by administration of a labeled or therapeutically-conjugated chemotactic peptide. In this application, the chemotactic peptides are chemically conjugated to DTPA and subsequently labeled with .sup.111 In. The utility of DTPA chelates covalently coupled to polypeptides and similar substances is well known in the art. Hnatowich, D. J., U.S. Pat. Nos. 4,479,930 and 4,668,503. Other bifunctional chelates for radiolabeling peptides, polypeptides and proteins are well known in the art. Other biologically active peptides described include that disclosed by Olexa S. A., Knight L. C. and Budzynski A. Z., U.S. Pat. No. 4,427,646, Use of Radiolabeled Peptide Derived From Crosslinked Fibrin to Locate Thrombi In Vivo, in which iodination is discussed as a means of radiolabeling. In Morgan C. A. Jr and Anderson D. C., U.S. Pat. No. 4,986,979, Imaging Tissue Sites of Inflammation, use of chelates and direct iodination is disclosed. In Tolman G. L., U.S. Pat. No. 4,732,864, Trace-Labeled Conjugates of Metallothionein and Target-Seeking Biologically Active Molecules, the use of metallothionein or metallothionein fragments conjugated to a biologically active molecule, including peptides, is disclosed. The previous methods all employ some conjugation means with a bifunctional chelator in order to effectuate labeling with a radionuclide or other medically useful metal ion, such as a paramagnetic contrast agent. The only exception involves radioiodination; the iodine labeling of proteins or peptides containing tyrosine or histidine residues is well known, for example, by the chloramine-T, iodine monochloride, Iodogen or lactoperoxidase methods.
The potential role of amino acid sequences found in peptides and proteins in binding transition metals has been recognized. In Vallee B. L. and Auld D. S.: Zinc coordination, function, and structure of zinc enzymes and other proteins, Biochemistry 29:5648-5659, 1990, the general characteristics of non-metallothionein proteins which contain zinc binding sites are described. Arnold F. H. and Haymore B. L. describe histidine-containing amino acid sequences used for protein purification by metal-chelate chromatography (Engineered metal-binding proteins: purification to protein folding, Science 252:1796-1797, 1991). Iverson et al. describe a means of genetic manipulation of antibodies to contain metal binding sites in the immunological binding region with the goal of producing catalytic antibodies (Iverson B. L., Iverson S. A., Roberts V. A., Getzoff E. D., Tainer J. A., Benkovic S. J. and Lerner R. A.: Metalloantibodies, Science 249:659-662, 1990). The use of histidine-containing amino acid sequences which bind Ru to form exchange-inert metal complexes to form highly stable .alpha.-helical metallopeptides was described in Ghardiri M. R. and Fernholz A. K.: Peptide architecture. Design of stable .alpha.-helical metallopeptides via a novel exchange-inert Ru.sup.III complex, J Am Chem Soc 112:9633-9635, 1990. The role of isolated amino acid ligands to bind .sup.99 Tc and .sup.99m Tc has long been recognized; Selfert et al. describes the capability of nitrogen donor atoms to stabilize reduced technetium species using free lysine, ornithine and histidine (Seifert S., Munze R. and Johannsen B.: Technetium-99 and 99 m chelates with N-donor ligands: a new class of potentially cationic radiopharmaceuticals, in Technetium in Chemistry and Nuclear Medicine Deutsch E., Nicolini M. and Wagner H. N. Jr, eds., Cortina International, Verona, 1983, pp 19-23. Similarly, use of free cysteine, cystine and penicillamine to bind .sup.99m Tc is known by those skilled in the art.